Ez c1 3

The Pt electrodes were covered with 400 μl of 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 7.5 buffer. The Pt wires were connected to an electric wave generator (TG330 function generator, Thurlby Thandar Instruments, Huntington, UK) under alternating current (AC) field conditions (500 Hz, 0.031 VRMS for 6 min; 500 Hz, 0.281 VRMS for 20 min, and 500 Hz, 0.623 VRMS for 1 h 30 min) at 37 °C. After GUV formation, the chamber was placed on an inverted confocal fluorescence microscope (Nikon D-ECLIPSE C1, Nikon, Melville, NY). The excitation wavelengths were 488 nm for ATG3-Alexa488 and 561 nm for Rho-PE. The images were collected using band-pass filters of 593 ± 20 nm for Rho-PE, and of 515 ± 15 nm for Alexa488. Then 1 μM ATG3-Alexa488 was added to study the ATG3 effect on the GUVs. All these experiments were performed at room temperature. Image treatment was performed using the EZ-C1 3.20 software (Nikon).

The Pt electrodes were covered with 400 μl of a 300 mM sucrose solution, previously equilibrated at 37 °C. The Pt electrodes were connected to a generator (TG330 function generator, Thurlby Thandar Instruments) under AC field conditions (10 Hz, 1 VRMS for 2 h, followed by 2.5 Hz, 1 VRMS, 1 h 30 min) at 37 °C. Finally, the AC field was turned off and the vesicles (in 300 mM sucrose) were collected from the PRETGUV 4 chamber with a pipette and transferred to chambers pretreated with bovine serum albumin (BSA) (2 mg/ml) and containing an equiosmolar buffer solution of 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 7.5. Due to the different density of the two solutions, the vesicles sedimented at the bottom of the chamber, and this facilitated observation under the microscope. Finally, ATG3-Alexa488 at 1 μM was added to study the ATG3 effect on the GUVs. The excitation wavelengths were 488 nm for ATG3-Alexa488 and 561 nm for Rho-PE; and the emission was collected using 515 ± 15 nm and 593 ± 20 nm band-pass filters, respectively. All these experiments were performed at room temperature. Image treatment was performed using the EZ-C1 3.20 software (Nikon).

The spectral confocal laser scanning microscopy (SCLSM) analysis of the stained samples was performed at 25 °C using the SCLSM system (Digital Eclipse C1si; Nikon) equipped with CFI S Fluor 4×, CFI Plan Apo10×, 20×, 40×, and VC60×H lenses and the EZ-C1 3.40 software (Nikon). Fluorescence was excited with the 408-nm line of a blue diode laser, the 488-nm line of a solid laser, and the 640-nm line of a diode laser. The emission spectra in the ranges of 460–470 nm, 500–650 nm, and 640–750 nm with 5 nm bandwidths were recorded for detecting the cell wall, TPI and lipid, and Alexa Fluor 647, respectively, with TPI and lipid simultaneously detected in the rage of 500–650 nm. Reference samples were prepared by dissolving commercial trans-polyisoprene in chloroform at a final concentration of 1 mg/mL, and thin films were prepared on glass slides. The films were stained with Nile red, and fluorescence spectra from each of 50 locations (regions of interest) were measured and averaged. The fluorescence spectra of the stained samples were obtained from 10 to 25 locations and averaged. Images were acquired and averaged from five successive scans to improve the signal-to-noise ratio. Image processing, including spectral unmixing, was performed using EZ-C1 3.40 software and Adobe Photoshop CS4.

All fusion constructs, GFP-EuTPTx, were generated by PCR methods using full-length EuTPTs and GFP as templates and ligated with the pBI121 vector. The resultant vectors were introduced into Agrobacterium tumefaciens LBA4404 by electroporation. Tobacco BY-2 cells stably expressing Golgi marker XYLT_CT-DsRed^{44 (link),45 (link)} were generated using pGPTV-HPT-XYLT_CT-DsRed and by Agrobacterium-mediated transformation^{45 (link)} and supertransformed with the GFP fusion constructs. Fluorescence signals were documented 3–4 days after the sub-cultivation. Cells expressing GFP and DsRed fusion proteins were first stained with 1 μM ER-tracker and analyzed with DIGITAL ECLIPSE C1si (Nikon, Tokyo, Japan) equipped with CFI Plan Apo objectives and EZ-C1 3.40 software (Nikon). Fluorescence was excited with the 408-nm line of a blue diode laser, the 488-nm line of a solid laser, and the 543-nm line of a G-HeNe laser.

For randomly chosen green fluorescent IN the largest cell diameter was determined manually in the confocal plane revealing the maximal soma shape by using EZ-C1 3.70 free viewer software (Nikon).